The risk factors of SARS-CoV-2 antibody level differences in healthcare workers post vaccination in Siloam hospitals: A nationwide multicenter study

Background Several vaccines have been approved against COVID-19, and 5 have been used in Indonesia. Due to the decrease in antibody levels 3 to 6 months after the second dose of CoronaVac, healthcare workers received the third booster of mRNA vaccine (mRNA-1273) to increase the antibody level. This study aimed to evaluate the risk factors of anti-S-RBD IgG levels differences in healthcare workers. Methods This study is a retrospective cohort study of 576 healthcare workers without previous SARS-CoV-2 infection who received 2 doses of CoronaVac and the third dose of mRNA-1273 6 months after the second dose. Blood samples were obtained 2nd, 6th, 12th, and 24th weeks after the second dose of CoronaVac vaccine administration, with mRNA-1273 booster on week 20. Quantitative measurements of IgG antibodies were performed with Elecsys Anti-SARS-CoV-2 S immunoassay. We identify the baseline factors predicting post-vaccination antibody titers using univariate and multivariate linear regression analysis. Results This study comprised 576 participants aged 32 years old, 72.05% female, and 45.84% from high-risk occupation subgroups. The median antibodies titer level on the 2nd, 6th, 12th, and 24th weeks after the second vaccine dose administration were 40.99 u/mL, 42.01 u/mL, 54.78 u/mL, and 23,225 u/mL. Antibody levels trended highest in female and younger age group (20-29 years old). Conclusions The third dose of vaccine increased the quantitative SARS-CoV-2 spike IgG antibody titers and eliminated differences in antibodies titer by gender.


Introduction
Within 2 years of the COVID-19 pandemic, COVID-19 vaccines were approved for use by the World Health Organization (WHO).As of 15 March, 2022 and 5 COVID-19 vaccines were used in Indonesia [1] .Vaccine efficacies (VE) ranged from 50% to 95% against symptomatic COVID-19 [2] .Studies assessing antibody levels in response to vaccination, however, have shown conflicting findings.Padoan et al. found that 6 months after the first BNT162b2 vaccine, antibody levels were not linked with age or gender but with previous COVID-19 infection [4] .These results contradict other studies that have reported age-and gender-dependence of antibody levels at 6 months [5][6][7][8] .
Reduced antibody levels have been linked to infections and transmissions, raising concerns about long-term protection to SARS-CoV-2 and prompting the possibility of a booster vaccine [4] .Antibody levels in healthcare professionals have been seen to drop between 3 and 6 months following the second vaccine dose, with the level at 6 months being comparable to those vaccinated with 1 dosage, suggesting a gradual weakening of immunological response over time [ 4 , 6 , 9 ].Antibody levels are reported to decrease earlier in the elderly and chronic renal disease, underweight, solid malignancy patients, and those on immunosuppressive medication, whereas they can increase in females [ 5 , 9 , 10 ].Interestingly, some studies revealed a small percentage of patients who had antibody levels 6 months after the initial dose that were labeled "late responders" rather than statistical outliers since they developed antibodies slowly after vaccination [ 4 , 9 ].The booster vaccine reduces the risk of symptomatic and asymptomatic illness, prevents transmission, and reduces viral load in infected unvaccinated people [11] .An earlier study found that the increase in antibody levels was significantly higher with heterologous regimens that included mRNA-based vaccines than with the homologous booster.Furthermore, the mRNA-1273 booster was found to be the most immunogenic and to have more reactogenicity than the BNT162b2 and Ad26.COV2.S boosters [12] .Few studies have examined booster vaccination antibody levels.
This study evaluated anti-S-RBD IgG level variations in healthcare professionals without previous SARS-CoV-2 infection following 2 doses of CoronaVac and mRNA-1273 booster 6 months after the second treatment.

Materials and methods
This retrospective cohort study was approved by Mochtar Riady Nanoinstitute for Nanotechnology (MRIN) ethical committee.The study was carried out between October 2021 and February 2022 in twelve branches of Siloam Hospitals throughout 8 out of 34 provinces in Indonesia.It included 576 healthcare professionals without previous SARS-CoV-2 infection who received 2 doses of CoronaVac (30 ug in 0.5mL of aluminum hydroxide diluent solution per dose) and a booster dose of mRNA-1273 (100 ug in 0.5 mL) 6 months after the second dose.Vaccines were injected into the deltoid muscle.Participants with a previous PCR swab test and regular anti-gen screening every 14 days were eliminated.COVID-19-infected patients with persisting anti-S1 IgG, anti-RBD total Ig, anti-S1 IgA, and SARS-CoV-2 neutralizing titers were eliminated [13] .
Antibodies were measured on the 2nd, 6th, 12th, and 24th weeks after the second dose of CoronaVac vaccine administration, with mRNA-1273 booster on the 20th week.This study evaluated anti-SARS-CoV-2 RBD antibodies using the FDA-recommended limit for seropositivity in convalescent plasma, 132 u/mL.The kit that was used in this study is Roche Elecsys Anti-SARS-CoV-2 S on Roche Cobas 6000 (Roche Diagnostics, Mannheim, Germany).In the 2nd and 6th weeks, antibody measurements were not diluted due to limited resources, yielding maximum results range of 250 u/mL.On the 12th and 24th weeks antibodies titer was diluted 400 times, yielding a maximum results range of 100.000 u/mL.On the 24th antibodies titer was measured after the third dose (booster) administration of the COVID-19 vaccine with mRNA Moderna vaccine during the 20th week.However, there were no baseline antibodies titer measured on the 20th week due to limited resources [14] .
Microsoft Excel summary data.SPSS for Windows was used for statistical analysis.Kolmogorov-Smirnov tested data normality.If p > 0.05, the data are normal.Normal data are displayed as mean ± SD, and abnormal distribution of data show as median (minimum-maximum).Categorical data are presented as frequency (percentage).The T-test and Mann-Whitney U-Whitney tests were used to evaluate continuous variables, and the  2 test was used to analyze categorical variables.If the data were not normally distributed, the Mann-Whitney or Kruskal Wallis tests would be used.The statistical significance was set to p < 0.05.The geometric mean titer of antibodies was calculated as the geometric mean of the positive serum samples.Multivariate analyses were performed to evaluate the correlation of antibody levels at the 2nd, 6th, 12th, and 24th weeks after the second dose of CoronaVac vaccine administration, with mRNA-1273 booster on the 20th week adjusted by age, gender, and occupational subgroups.

Results
The study was carried out between October 2021 and February 2022 in twelve branches of Siloam Hospitals throughout 8 out of 34 provinces in Indonesia.This study comprised 576 health care professionals who had received 3 doses of the vaccination and who had never been infected with COVID-19.The mean age was 31.97 ± 8.29.Most participants (72.05%) were female and worked in high-risk occupations such doctors, nurses, midwives, and pharmacists.( Table 1 ).
Antibodies were measured on the 2nd, 6th, 12th, and 24th weeks after the second dose of CoronaVac vaccine,   2 and Figure 1 .Differences between antibodies titer levels between the 2nd, 6th, 12th, and 24th weeks measurement with the Mc-Nemar test are shown in Table 3 .The results were considered low level was < 132 u/mL, and high-level was ≥ 132 u/mL.During the 6th week, 5.77% of participants who previously had low-level on the 2nd week developed an increasement to high-level.The remaining 72.22% with high level on the 2nd week ( p -value 0.79), continued to have high level.On the 12th week, 16.35% with low level developed an increasement, and 65.72% with high level antibody on the 6th week continued to have high-level with a p -value < 0.001.On the 24th week, 97.86% developed high level, and 100% who had high-level on the 12th week continued to have high-level with a p -value < 0.001.( Table 3 ).
As shown in Table 4 , 5 , 6 and Figure 2 , median antibody titer levels were compared by age, gender, and profession subgroups.Median antibody titer levels in 2nd and 6th weeks were significantly higher in the younger age group ( < 50 years old) than in the older age group ( ≥ 50 years old), with a p -value of 0.042 and 0.044.On the 12th and 24th weeks, there were no significant median antibody titer levels differences between each group, with a p -value of 0.064 and 0.183.On the 2nd, 6th, and 12th weeks, median antibody titer levels were significantly higher in the female group with a p -value of 0.03, < 0.001, < 0.001.On week 24, the median antibody titer levels for male and female subgroups were 24,109 and 22,911 u/mL, respectively, with a p -value of 0.52 indicating no significant difference.On the 2nd, 6th, 12th, and 24th weeks, antibody titer levels in highrisk and low-risk occupational subgroups were not significantly different, with a p -value of 0.52, 0.46, 0.39, and 0.06.
Multivariate linear regression of age showed significant regression with antibody titer level (log) on 2nd week.Age and gender have significant regression with antibody titer level on 6th week.Only age has showed significant regression with antibody titer level on 12th week.Among the 24th week after second dose, antibody titer level showed no significant regression with age, gender, and high-risk occupation ( Table 7 ).

Discussions
The results showed no significant difference between antibody titer levels during week 2 and 6 measurements, which might be affected by no dilution measurement procedure.Median antibody titers were significantly different on the 2nd, 6th, and 12th weeks.There was a significant difference between median antibody titer levels on  This study showed antibody titer levels were significantly higher in the younger age group than the older age group on the 2nd and 6th weeks.However, there was no significant difference on the 12th and 24th weeks.Decreases  in antibody titer level on the 12th week in younger and older age groups suggest the need of booster dose.These findings are similar to those produced by Shrotri et al [16] , which showed waning of antibody level over 3-10 weeks after second vaccination dose, hence booster dose was suggested to be administered within 8-12 weeks after vaccination.Age was dominant factor in multivariate analysis that inversely associated with antibody titer levels.The findings are in accordance with Terpos et al. [17] in Greece reported an age-dependent pattern of immune responses less prominent in the elderly.Müller et al. [18] demonstrated that antibody titer levels were significantly lower in the elderly than in the young subject.Immune function declines with age, known as immunosenescence.In the elderly, the activity of natural killer cells and dendritic cells are reduced, and decreased variety and memory of T and B cells are seen in both the innate and the adaptive immune systems [19] .The response before the subsequent measurement could significantly predict antibody titer levels in this study.Antibody titer levels were also observed to be considerably greater in the female group compared to the male group on the 2nd, 6th, and 12th weeks.However, there were no significant differences on the 24th week.This result is stated by recent data published by Salvagno et al. [20] , who also found that females had a significantly higher response of total antibody titer level after the second dose of mRNA BNT162b2.Males with COVID-19 had a greater risk of in-hospital intubation, 3 times the odds of ICU admission, and a longer hospital stay [ 21 , 22 ].Several explanations exist.Females have more CD4 + T cells, stronger CD8 + T cell cytotoxic activity, and more immunoglobulin-producing B cells than males.Females get more severe local and systemic side effects from immunizations and create larger antibody titers.Therefore, these imply that females can mount humoral immune responses more than males.Females generate more type 1 interferon (IFN), a strong anti-viral cytokine, when tolllike receptor 7 senses viral RNA than males, which is crucial for the early response to COVID-19.The X chromosome, which carries immune-related genes, is linked to increased IFN production in females [23] .Thirdly, estradiol (E2) offers an advantage against infectious disease by augmenting T cell responses and increasing antibody production, neutrophils, and monocyte/macrophage cytokine production [ 21 , 22 ].Therefore, menopause is an independent risk factor for female COVID-19 patients and is associated with more severe COVID-19 due to decreased E2 [24] .In contrast, testosterone is associated with immune system suppression, age-related decline in B cells, and a trend towards accelerated immune aging in males [ 20 , 21 ].These also could explain why the prevalence of autoimmune diseases is more remarkable in females with a ratio of 2:1 [23] .The lower antibodies titer level in males and older samples would suggest that this specific population may have less efficient protection against infection and an even higher risk of developing more severe COVID-19 infection.Therefore, it would be advisable for this specific population to receive a timely second vaccine dose and even a booster dose.The gender group and log antibody on 2nd and 6th weeks after second dose administration differed significantly.However, there was no significant difference on the 12th and 24th weeks.The au-thors suggested that the third dose of vaccine eliminates differences in antibody titer levels by gender.
Terpos et al. [17] and Levin et al. [5] reported antibody titer level decreases significantly within 4 weeks up to 6 months after the second dose of vaccine administration.However, in this study, there was a significant increase in antibody titer level measured on the 24th week, contrary to previous studies.On the 24th week antibody titer level was evaluated following mRNA-1273 booster vaccination delivery on the 20th week, therefore it is unknown if the antibody titer declined, maintained, or grew before the booster.Therefore, further studies with baseline antibodies titer measurement are recommended 6 months after the second vaccination dose and after the booster dose.No significant difference in antibody titer levels on the 2nd, 6th, 12th, and 24th weeks between the occupational group at high risk of COVID-19 transmission and low-risk groups.
The first limitation of the study is a technical problem in antibody titer that is not using dilution in the measurement process due to limited resources.Therefore, this study use the median rather than the mean to reduce the bias risk.The second limitation is the lack of baseline antibodies before the mRNA-1273 booster vaccine due to limited resources.Therefore, there was a significant increase in antibodies titer measured on the 24th week.The third limitation is that this study only uses age, gender, and occupation as the independent variable, which might lead to potential bias due to limited data to be analyzed.

Conclusion
Overall, post-COVID-19 vaccine humoral response is associated with 3 essential predictors, age, sex, and baseline serostatus.Therefore, identifying populations with a risk of inadequate post-vaccine immune response will be necessary to prevent COVID-19 infections and mortality and limit the potential of emerging SARS-CoV-2 mutation variants.Booster vaccination increased antibody levels, hence boosting with any available vaccine was better than not boosting to minimize infection and transmission.Booster protection is needed during the COVID-19 surge, as this variant seems to infect vaccinated persons.Booster immunization increases antibody titer levels, adding protection.Further investigations using baseline antibody titer measurements 6 months after the second dose of immunization and after the booster dose are recommended.

Funding
Antibody test kit was funded by Roche Indonesia.The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Table 1
Description demographic data of participants.

Table 2
Serum antibody titer measured on 2nd, 6th, 12th, and 24th weeks after second dose of vaccination.

Table 4
Antibody titer levels by age subgroups.

Table 5
Antibody titer levels by gender subgroups.
that ofTakeuchi et al., however in this study, a significantly bigger sample size was used and it was obtained from healthcare facilities located in multiple locations.

Table 6
Antibody titer levels by occupation subgroups.

Table 7
Multivariate linear regression of age, gender, occupation compared with antibody titer levels.
* Transformation data of Log Antibody.